Registration Dossier

Administrative data

Endpoint:
three-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1978
Report Date:
1978

Materials and methods

Principles of method if other than guideline:
A three-generation study was conducted in CD rats with 20 males and 20 females per group. Each group received the test material by dietary administration. Observations were made to determine litter size, litter and pup weights, pup mortality, and gross abnormalities. Gross and histopathological observations were conducted on rats of the F3b generation.

GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: not reported

Test animals

Species:
rat
Strain:
other: CD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Manston, Kent, England.
- Age at study initiation: not reported
- Weight at study initiation: (F0) Males: 76 g; Females: 72 g; (F1) Males: 122 - 130 g; Females: 107 - 113 g; (F2) Males: 115 - 130 g; Females: 107 - 119 g
- Housing: During the pre-mating period the rats were housed five to a cage with cages of males interspersed among those holding females to promote the development of regular oestrus cycles. After mating males were returned to their original cages, whilst the females were transferred to individual cages (equipped with solid floors) for the birth and rearing of litters.

- Diet: ad libitum (Spratt's Laboratory Diet No. 2)
- Water: ad libitum
- Acclimation period: seven days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 50 ± 5%
- Air changes (per hr): 12 - 14


Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
The test compounds were administered in the diet (Spratt's Laboratory Diet No. 2) and fresh batches of diet were mixed weekly. Treatment continued throughout the study for all generations. Concentrations of greater than 5% were attained by weekly increments following a 5, I0 and 20% sequence to achieve appropriate concentrations with reciprocal reduction of rice starch
Details on mating procedure:
- M/F ratio per cage: 1 to 1
- Length of cohabitation: 14 days
- Mating for second litter: Shortly following (approximately 10 days) sacrifice of the first litters, the animals were re-mated (for a period of 19 days) ensuring that different males and females were coupled within each group.
- Proof of pregnancy: Daily vaginal smears were taken during the mating period and the day on which spermatozoa were present (or a copulation plug found) was considered day 0 of pregnancy.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
A control group received 20% rice starch, and a comparison group 20% sucrose ad libitum.
Graded diets of test substance, 2, 5, 10 or 20% were fed to treatment groups with rice starch q.s. to bring the level to 20%. For test substance groups receiving diets greater than 5%, administration began at the 5% level with 5% weekly increments until final doses of 5, 10, 15 or 20% were reached.

During parturition, the diets were reduced to 5% test substance.
After parturition of second litters of each generation, dams receiving diets greater than 5% were returned to this level.

Males and non-pregnant females received full dose.
After weaning, the dose administered to F1B and F2B generations were progressively increased until full treatment level was attained.
Frequency of treatment:
Daily in feed
Details on study schedule:
- Selection of parents: Once animals of the F0 generation (and, subsequently, weanlings of the F1B and F2B generations) had been acclimatised to their full dietary concentrations they were maintained on their respective diets until females had attained 180/200 g weight (or were at least 9 weeks old). The animals were then mated on a one male to one female basis for a minimum period of 14 days, subsequently, males were returned to their original cages, whilst the females were transferred to individual cages (equipped with solid floors) for the birth and rearing of litters. Offspring of this pairing were sacrificed on day 4 post partum.

Shortly following (approximately 10 days) sacrifice of the first litters, the animals were re-mated (for a period of 19 days) ensuring that different males and females were coupled within each group. In the first generation, all offspring of the second pairing were reared to 21 days post partum. However, in an attempt to overcome the high, but non-selective, pup mortality which occurred between days 4 and 12, agreement was reached with our sponsors to cull second litters of the subsequent F1B and F2B generations on day 4 post partum to give an equalised litter size of eight, where possible.

From the second litters of the initial (F0) and second (F1B) generations, twenty males and twenty females were selected from each group at weaning to form the basis of the second and third (F2B) generations, respectively. The animals were selected from as many litters as practical, derived from
the first two weeks' mating, and as close to the mean weaning weight for each sex as possible. Brother and sister matings were avoided for the second and third generations.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 2, 5, 10 and 20%
Basis:
nominal in diet
No. of animals per sex per dose:
Twenty male and female rats from each dose group
Control animals:
yes, plain diet
Positive control:
Rats were tested with 20% Sorbitol and 20% Sucrose

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were regularly handled and observed daily for obvious changes or signs of reaction to treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Any rat which showed marked signs of ill health or reaction to treatment was isolated and/or killed to prevent cannibalism or autolytic degeneration. All animals that died or were killed for humane reasons were weighed and subjected to post mortem examination.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat of the F0 generation was determined initially and subsequently at weekly intervals up to pairing. Animals of the F1B generation were weighed at birth, 4, 12 and 21 days post partum and subsequently at intervals of one week to pairing. For the F2B generation, pups were weighed, additionally, on day 8 post partum.
During the mating periods, females were also weighed on alternate days until the occurrence of a positive indication of mating (i.e. sperm or plug): thereafter they were weighed on presumed days 0, 7, 14 and 20 of gestation. Weights of pregnant animals without positive indication of mating were calculated retrospectively from birth assuming a 22 day gestation period. Dams were weighed on days 0, 7, 14 and 21 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption: Yes; Food intake of rats was recorded weekly during the first pre-mating period of each generation (F0, F1B, F2B).

OTHER:
PREGNANCY RATE: Pregnancy rate was determined as the percentage of surviving paired females that became pregnant.

MATING PERFORMANCE: Daily vaginal smears were taken during the mating period and the day on which spermatozoa were present (or a copulation plug found) was considered day 0 of pregnancy.
Oestrous cyclicity (parental animals):
Estrous cycle length and normality were evaluated in F0 and F1 females by vaginal smears prior to mating, and during mating until evidence of mating was found.
Litter observations:
LITTER OBSERVATIONS: As soon as possible after parturition, which was observed wherever possible, the young were counted (for numbers of live and dead), weighed individually and examined for external abnormalities. Keeping nest disturbance to a minimum, all litters were examined daily for dead and abnormal young.

First Litters (F1A, F2A, F3A):
Young of the F1A generation were weighed individually on day 4 post partum and sex was determined by external inspection of the ano-genital distance. Young were then sacrificed and examined externally and internally for abnormalities; sex of the pups was confirmed by gonadal inspection. Where indicated, pups were preserved in the appropriate fixative for further examination.

Second litters (F1B, F2B, F3B):
As soon as possible after birth, young of the F1B generation were individually identified within the litter (toe marked) and weighed on days 4, 12 and 21 post partum.
Young of the F2B and F3B generations were weighed and sexed at day 4 post partum when the litter size was reduced to 4 males and 4 females (total 8) except for occasional instances where this was made impractical by the original constitution of the litter. Reduction of the litter size was achieved by selecting for continuation 4 males and 4 females as close to the mean weight of each sex in the litter, the surplus males and
females were then killed and examined.
Young were weighed on days 8, 12 and 21 post partum. Surplus pups of the F1B and F2B generations were sacrificed on, or shortly after, day 21 and examined externally and internally for abnormalities; sex of the pups was confirmed by gonadal inspection.
Postmortem examinations (parental animals):
After the second litters had been weaned, parent animals of each generation (F0, F1B, F2B) were sacrificed and examined macroscopically.
Postmortem examinations (offspring):
- Litter size:
At birth, mean values were determined both for the total number of young and for the number of viable young.

- Litter and mean pup weights:
Litter weight was calculated from the individual pup weights.

- Pup mortality:
Pup mortality rates were calculated as % losses within individual litters at birth and cumulatively on days 4, (8), 12 and 21 (for equalised litters of the F1A generation, cumulative loss was calculated from day 4). Group mean values were calculated from individual litter percentages.

- Abormalities:
AIl dead young, (except those excessively cannibalised) were autopsied. Pups with suspected abnormalities were preserved either in Bouin's solution if it was considered that free-hand sectioning (Wilson technique) or histopathological examination would be of value, or in alcohol for subsequent staining with alizarin (modified Dawson technique) if skeletal defects were suspected.

- Histopathology of F3B generation:
See Table 1 below
Statistics:
Litter data are statistically analysed on a litter basis using non-pametric methods. Absolute and relative organ weights were subjected to analysis of variance. Analysis of covariance was also carried out adjusting for bodyweight. Williams test for comparing increasing doses of a compound with a control was used for Xylitol groups and also, independently, for Sorbitol and Sucrose against the control group.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
See "Any other information on Results"

ORGAN WEIGHTS (PARENTAL ANIMALS):
See "Any other information on Results"

GROSS PATHOLOGY (PARENTAL ANIMALS):
During terminal sacrifice of F2B parents caecal enlargement appeared to be occurring in some groups and thereafter the weight of the caecum and contents of remaining dams and of ten males per group was determined. Although visual examination did not always indicate overt caecal enlargement, the weighing showed on increased caecal weight among Sorbitol fed animals and a dosage related increase in caecal weight for test substance fed animals, the effect being greater among females than males. Except for both sexes, fed 2% test substance and males fed 10% test substance intergroup differences from control values were statistically significant

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
no NOAEL identified Generation not specified (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

VIABILITY (OFFSPRING):
See "Any other information on Results"

BODY WEIGHT (OFFSPRING):
See "Any other information on Results"

ORGAN WEIGHTS (OFFSPRING):
See "Any other information on Results"

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

At low diet levels (2 and 5%) food intake was comparable with controls in all generations. At 10% test substance, food intake was slightly but consistently lower in the second generation. At the 20% level, marked suppression of food intake in F1and F2 generations was seen, even when test substance concentration was reduced to 5%. With this group consistent but generally marked suppression of food intake was noted in all generations even at the lowest level fed. With the sucrose group food intake was comparable with or slightly greater than that of the control group in all generations. At the lower levels, weight gain of males of the first generation (2, 5, or 10%) was comparable to the control group, slight suppression occurred at the 10% level during the second generation, and at all concentrations in the final generation (F3b). At the highest level (20%) a larger proportion of litters were born on day 23 or 24 compared to controls, or sucrose. Caecal enlargement was noted at terminal necropsy of F2b parents of both sexes for levels greater than 2%.

 

At the 20% dietary level of test substance there were lower values for total and viable litter size at birth and at day four postpartum. This became more accentuated with successive generations. Litter and mean pup weights were greater than concurrent control values except for 20% test substance at which level lower litter weights reflected smaller litter size. Initially among the F1a pups, when litter size was not culled, a large mortality was noted between seven to 10 days postpartum. The litter size was culled to eight with the succeeding generations, at which time no total litter loss was observed. There was no indication of a treatment effect on occurrence of terata. Statistical assessment of organ weight data showed in most instances changes related to differences in body weight. Statistically significant lower absolute thyroid weights were noted with 20% test substance. Other organ weight changes appeared sporadically, unrelated to treatment. Histopathologically tissues from 10 and 20% test substance groups did not show microscopic changes that could be attributed to test substance.

Applicant's summary and conclusion

Conclusions:
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
Executive summary:

A three-generation study was conducted in CD rats with 20 males and 20 females per group. Each group received the test material by dietary administration. Observations were made to determine litter size, litter and pup weights, pup mortality, and gross abnormalities. Gross and histopathological observations were conducted on rats of the F3b generation.

Mortalities of parents were randomly distributed. No obvious clinical signs were noted. At low diet levels (2 and 5%) food intake was comparable with controls in all generations. At 10% test substance, food intake was slightly but consistently lower in the second generation. At the 20% level, slight depression of food intake of the F0, and marked suppression in F1and F2 generations was seen, even when test substance concentration was reduced to 5%. With this group consistent but generally marked suppression of food intake was noted in all generations even at the lowest level fed. With the sucrose group food intake was comparable with or slightly greater than that of the control group in all generations. At the lower levels, weight gain of males of the first generation (2, 5, or 10%) was comparable to the control group, slight suppression occurred at the 10% level during the second generation, and at all concentrations in the final generation (F3b). There was no treatment effect on mating performance and pregnancy rate. At the highest level (20%) a larger proportion of litters were born on day 23 or 24 compared to controls, or sucrose. Caecal enlargement was noted at terminal necropsy of F2b parents of both sexes for levels greater than 2%. At the 20% dietary level of test substance there were lower values for total and viable litter size at birth and at day four postpartum. This became more accentuated with successive generations. Litter and mean pup weights were greater than concurrent control values except for 20% test substance at which level lower litter weights reflected smaller litter size. Initially among the F1a pups, when litter size was not culled, a large mortality was noted between seven to 10 days postpartum. The litter size was culled to eight with the succeeding generations, at which time no total litter loss was observed. There was no indication of a treatment effect on occurrence of terata. Statistical assessment of organ weight data showed in most instances changes related to differences in body weight. Statistically significant lower absolute thyroid weights were noted with 20% test substance. Other organ weight changes appeared sporadically, unrelated to treatment. Histopathologically tissues from 10 and 20% test substance groups did not show microscopic changes that could be attributed to test substance.